Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.17 (lysozyme)
21,489 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Interactions of several proteins with glutathione-insulin transhydrogenase (GIT) have been investigated by determining their ability to inhibit degradation of 125I-labeled insulin catalyzed by GIT. The inhibition by every insulin analog (des-Asn-des-Ala-pork insulin, desoctapeptide-pork insulin, des-Ala-pork insulin, pork insulin, proinsulin, and guinea pig insulin) was competitive vs. competitive vs. insulin indicating that they function as alternate substrates. The insulin analogs with the least hormonal activity showed the highest potency as inhigitors of insulin degradation. Whereas native ribonuclease and lysozyme showed little or no inhibition, their scrambled forms (i.e. reduced and randomly reoxidized) showed competitive inhibition with a potency greater than that of insulin. These results suggest that the conformation of the substrate or inhibitor is probably the major factor in determining the specificity for (or binding to) the enzyme. Studies withother peptide hormones showed competitive inhibition with vasopressin and oxytocin and noncompetitive inhibition with glycagon. The inhibition with growth hormone could be either competitive or noncompetitive. The inhibition by glucagon and growth hormone (physiologic antagonists of insulin) could serve as a control mechanism to modulate the activity of enzyme. The following showed very little or no inhibition; the native and scrambled form of pepsinogen, trypsin inhibitor of beef pancreas and of lima bean, C-peptide of pork proinsulin, and heptapeptide (B23-B29) of insulin.
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PMID:Interaction of insulin analogs, glucagon, growth hormone, vasopressin, oxytocin, and scrambled forms of ribonuclease and lysozyme with glytathione-insulin transhydrogenase (thiol: protein-disulfide oxidoreductase): dependence upon conformation. 117 Aug 77

Under hydrolytic conditions using 6 M HCl, tryptophan reacted separately with dithiodiglycolic acid and cystine to give beta-3-oxindolylalanine (beta-[3-(2-indolinone)]alanine) as the main product. A compound, which eluted in the amino acid analyzer at the same position as beta-3-oxindolylalanine, was found in the acid hydrolyzate of lysozyme. The identicalness of these two compounds was established by comparison of their ultraviolet absorption spectra, elution positions on ion exchange chromatograms, etc. The "acid degradation product of tryptophan", which is known to be produced upon acid hydrolysis of tryptophan-containing proteins, must also be the same compound.
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PMID:Beta-3-Oxindolylalanine: The main intermediate in tryptophan degradation occurring in acid hydrolysis of protein. 125 56

The lysozyme from bacteriophage T4 is being used as a model system to determine the roles of individual amino acids in the folding and stability of a typical globular protein. One general finding is that the protein is very adaptable, being able to accommodate many potentially destabilizing replacements. In order to determine the importance of 'alpha-helix propensity' in protein stability, different replacements have been made within alpha-helical segments of T4 lysozyme. Several such substitutions of the form Xaa-->Ala increase the stability of the protein, supporting the idea that alanine is a strongly helix-favouring amino acid. It is possible to engineer a protein that has up to ten alanines in succession, yet still folds and has normal activity. This illustrates the redundancy that is present in the amino acid sequence. A number of 'cavity-creating' mutants of the form Leu-->Ala have been constructed to understand better the nature of hydrophobic stabilization. The structural consequences of these mutations differ from site to site. In some cases the protein structure hardly changes at all; in other cases removal of the wild-type side-chain allows surrounding atoms to move in and occupy the vacated space, although a cavity always remains. The destabilization of the protein associated with these cavity-creating mutations also varies from case to case. The results suggest how to reconcile recent conflicting reports concerning the strength of the hydrophobic effect in proteins.
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PMID:Dissection of protein structure and folding by directed mutagenesis. 129 Sep 31

The addition of cationic proteins such as lysozyme, ribonuclease and cytochrome C enhanced the beta-lactam-induced bacteriolysis of staphylococci measured as release of wall label or by optical density. The treatment of staphylococci with penicillin plus cytochrome C resulted in a reduced viability of bacteria compared with those treated with penicillin alone. The wall autolysis and the penicillin-induced bacteriolysis of staphylococci were enhanced by the lysosomal enzyme cathepsin C. The penicillin-induced bacteriolysis was also enhanced by the D-amino acids D-alanine and D-methionine, while the comparable L-amino acids did not reveal any activity. On the other hand, some polyanionic substances were able to suppress the penicillin-induced bacteriolysis. Radiochemical and electron microscopic studies revealed the participation of bacterial wall autolysins in the first steps of degradation processes of staphylococcal walls within murine bone marrow-derived macrophages.
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PMID:The modulation of the bacteriolytic effect of beta-lactam antibiotics by non-antibiotics. 129 43

In a systematic attempt to identify residues important in the folding and stability of T4 lysozyme, five amino acids within alpha-helix 126-134 were substituted by alanine, either singly or in selected combinations. Together with three alanines already present in the wild-type structure this provided a set of mutant proteins with up to eight alanines in sequence. All the variants behaved normally, suggesting that the majority of residues in the alpha-helix are nonessential for the folding of T4 lysozyme. Of the five individual alanine substitutions it is inferred that four result in slightly increased protein stability and one, the replacement of a buried leucine with alanine, substantially decreased stability. The results support the idea that alanine is a residue of high helix propensity. The change in protein stability observed for each of the multiple mutants is approximately equal to the sum of the energies associated with each of the constituent substitutions. All of the variants could be crystallized isomorphously with wild-type lysozyme, and, with one trivial exception, their structures were determined at high resolution. Substitution of the largely solvent-exposed residues Asp 127, Glu 128, and Val 131 with alanine caused essentially no change in structure except at the immediate site of replacement. Substitutions of the partially buried Asn 132 and the buried Leu 133 with alanine were associated with modest (< or = 0.4 A) structural adjustments. The structural changes seen in the multiple mutants were essentially a combination of those seen in the constituent single replacements. The different replacements therefore act essentially independently not only so far as changes in energy are concerned but also in their effect on structure. The destabilizing replacement Leu 133-->Ala made alpha-helix 126-134 somewhat less regular. Incorporation of additional alanine replacements tended to make the helix more uniform. For the penta-alanine variant a distinct change occurred in a crystal-packing contact, and the "hinge-bending angle" between the amino- and carboxy-terminal domains changed by 3.6 degrees. This tends to confirm that such hinge-bending in T4 lysozyme is a low-energy conformational change.
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PMID:Multiple alanine replacements within alpha-helix 126-134 of T4 lysozyme have independent, additive effects on both structure and stability. 130 17

The functional role of tyrosine-63 in the catalytic action of human lysozyme (EC 3.2.1.17) has been probed by site-directed mutagenesis. In order to identify the role of Tyr63 in the interaction with substrate, both the three-dimensional structures and the enzymatic functions of the mutants, in which Tyr63 was converted to phenylalanine, tryptophan, leucine, or alanine, have been characterized in comparison with those of the wild-type enzyme. X-ray crystallographical analysis of the mutant enzyme at not less than 1.77-A resolution indicated no remarkable change in tertiary structure except the side chain of 63rd residue. The conversion of Tyr63 to Phe or Trp did not change the enzymatic properties against the noncharged substrate (or substrate analogs) largely, while the conversion to Leu or Ala markedly reduced the catalytic activity to a few percent of wild-type enzyme. Kinetic analysis using p-nitrophenyl penta-N-acetyl-beta-(1----4)-chitopentaoside (PNP-(GlcNAc)5) as a substrate revealed that the reduction of activity should mainly be attributed to the reduction of affinity between enzyme and substrate. The apparent contribution of the phenolic hydroxyl group and the phenol group in the side chain of Tyr63 was estimated to 0.4 +/- 0.4 and 2.5 +/- 0.8 kcal mol-1, respectively. The result suggested that the direct contact between the planar side-chain group of Tyr63 and the sugar residue at subsite B is a major determinant of binding specificity toward a electrostatically neutral substrate in the catalytic action of human lysozyme.
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PMID:Dissection of the functional role of structural elements of tyrosine-63 in the catalytic action of human lysozyme. 139 Jul 8

Pepsin successfully catalyzed the synthesis of several peptide derivatives from N-protected di- or tripeptides and amino acid or peptide esters or p-nitroanilides in dimethylformamide-water solutions at pH 4.6. An optimal substrates:pepsin ratio depended on the structure of starting peptides, especially their fit to the substrate binding sites of the enzyme. For hexapeptide Z-Ala-Ala-Phe-Leu-Ala-Ala-OCH3 formation, an equilibrium yield was attained at 1:3.10(5) enzyme-substrates ratio that indicated high efficiency of pepsin in synthesis reactions. In the course of the equilibrium peptide synthesis, pepsin gradually disappeared from the liquid phase due to its entrapment within a gel, formed by the hexapeptide product, while retaining its activity. The inclusion into the precipitate was not specific for pepsin, so far as inert proteins, lysozyme, ribonuclease A and carbonic anhydrase, when added to the reaction mixture, became also co-precipitated with the hexapeptide formed. It appears that co-precipitation of pepsin, an important factor limiting the enzyme efficiency, might be operative as well for other proteinases used to catalyze peptide synthesis.
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PMID:Pepsin as a catalyst of peptide synthesis. Enzyme co-precipitation with emerging peptide products. 142 33

It was previously shown that the two replacements Gly 77-->Ala (G77A) and Ala 82-->Pro (A82P) increase the thermostability of phage T4 lysozyme at pH 6.5. Such replacements are presumed to restrict the degrees of freedom of the unfolded protein and so decrease the entropy of unfolding [B. W. Matthews, H. Nicholson, and W. J. Becktel (1987) Proceedings of the National Academy of Science USA Vol. 84, pp. 6663-6667]. To further test this approach, three additional replacements--G113A, K60P and A93P--have been constructed. On the basis of model building, each of these three replacements was judged to be less than optimal because it would tend to introduce unfavorable van der Waals contacts with neighboring parts of the protein. The presence of such contacts was verified for G113A and K60P by conformational adjustments seen in the crystal structures of these mutant proteins. In the case of G113A there are backbone conformational changes of 0.5-1.0 A in the short alpha-helix, 108-113, that includes the site of substitution. In the case of K60P the pyrrolidine ring shows evidence of strain. The thermal stability of each of the three variants at both pH 2.0 and pH 6.5 was found to be very close to that of wild-type lysozyme. The results suggest that the procedure used to predict sites for both Xaa-->Pro and Gly-->Ala is, in principle, correct. At the same time, the increase in stability expected from substitutions of this type is modest, and can easily be offset by strain associated with introduction of the alanine or proline. This means that the criteria used to select substitutions that will increase thermostability have to be stringent at least. In the case of T4 lysozyme this severely limits the number of sites. The analysis reveals a significant discrepancy between the conformational energy surface predicted for the residue preceding a proline and the conformations observed in crystal structures.
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PMID:Analysis of the effectiveness of proline substitutions and glycine replacements in increasing the stability of phage T4 lysozyme. 145 24

A mutant human lysozyme C77/95A, in which Cys77 and Cys95 are replaced with alanine, has been characterized by 8-fold greater secretion in yeast (Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M., and Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967) and almost the same three-dimensional structure as wild-type human lysozyme (Inaka, K., Taniyama, Y., Kikuchi, M., Morikawa, K., and Matsushima, M. (1991) J. Biol. Chem. 266, 12599-12603). To clarify the molecular features of C77/95A and the reason for its increased secretion in yeast, the stabilities of the mutant C77/95A and the wild-type proteins were examined by guanidine hydrochloride denaturation, and the unfolding-refolding kinetics were determined from circular dichroism and fluorescence stopped-flow measurements. Equilibrium experiments showed that the delta G of unfolding of C77/95A in water was 5.8 kcal/mol less stable than that of the wild-type protein at pH 4.0 and 10 degrees C. The unfolding rate of C77/95A was 4 orders of magnitude faster than that of the wild-type protein whereas the two proteins shared similar refolding rates. The slowly refolding phase of the wild-type protein disappeared in C77/95A, indicating that the disulfide bond affects this phase. These observations show that the disulfide bond Cys77-Cys95 contributes to the stabilization of the folded form of human lysozyme by suppressing the unfolding rate and that the increase in the unfolding rate, or the disappearance of the slowly refolding phase in vitro, could correlate with the increase in secretion efficiency in vivo.
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PMID:Folding mechanism of mutant human lysozyme C77/95A with increased secretion efficiency in yeast. 153 44

Six "cavity-creating" mutants, Leu46----Ala (L46A), L99A, L118A, L121A, L133A, and Phe153----Ala (F153A), were constructed within the hydrophobic core of phage T4 lysozyme. The substitutions decreased the stability of the protein at pH 3.0 by different amounts, ranging from 2.7 kilocalories per mole (kcal mol-1) for L46A and L121A to 5.0 kcal mol-1 for L99A. The double mutant L99A/F153A was also constructed and decreased in stability by 8.3 kcal mol-1. The x-ray structures of all of the variants were determined at high resolution. In every case, removal of the wild-type side chain allowed some of the surrounding atoms to move toward the vacated space but a cavity always remained, which ranged in volume from 24 cubic angstroms (A3) for L46A to 150 A3 for L99A. No solvent molecules were observed in any of these cavities. The destabilization of the mutant Leu----Ala proteins relative to wild type can be approximated by a constant term (approximately 2.0 kcal mol-1) plus a term that increases in proportion to the size of the cavity. The constant term is approximately equal to the transfer free energy of leucine relative to alanine as determined from partitioning between aqueous and organic solvents. The energy term that increases with the size of the cavity can be expressed either in terms of the cavity volume (24 to 33 cal mol-1 A-3) or in terms of the cavity surface area (20 cal mol-1 A-2). The results suggest how to reconcile a number of conflicting reports concerning the strength of the hydrophobic effect in proteins.
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PMID:Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect. 155 43


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